Real-time two-step RT-PCR was carried out using tenfold dilutions of human leukocyte cDNA (10 ng to 0.1 ng) and a QuantiTect Primer Assay for BCL2 (B-cell CLL/lymphoma 2). Reactions were run in triplicate using either [A] the Rotor-Gene SYBR® Green PCR Kit and Rotor-Gene Q or [B] a kit and cycler from Supplier AII. The Rotor-Gene system provided lower CT values and greater reproducibility within triplicates. Melting curve analysis (see insets) showed a single peak, demonstrating specific amplification of the target.|Real-time PCR was carried out using tenfold dilutions of plasmid DNA (109 to 10 copies) and a QuantiTect Primer Assay for PPIA (cyclophilin A). Reactions were run in triplicate using the Rotor-Gene SYBR® Green PCR Kit and Rotor-Gene Q. [A] The Rotor-Gene system provided reliable detection over the entire range of template dilutions as well as highly reproducible CT values within each set of triplicates. [B] Melting curve analysis indicates specific amplification of the target.|Cations in the Rotor-Gene Q PCR buffer increase specific primer annealing. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing. NH4+ destabilizes weak hydrogen bonds between mismatched bases.|[A] Q-Bond in Rotor-Gene SYBR® Green PCR Master Mix increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|